Fish pancreatic disease virus

ABSTRACT

There is described a novel virus which is the causative agent of fish pancreatic disease, which is a serious disease affecting Atlantic salmon. A method of isolating the virus through co-cultivation of infected tissues following by passage is described and an isolated strain of the virus has been deposited. The use of the virus, or proteins or polypeptide derived therefrom as a vaccine or in diagnosis is described, together with genetically engineered constructs.

FIELD OF THE INVENTION

There is provided a novel virus which is a causative agent of fishpancreas disease. The virus may be used to provide a vaccine and/or amethod of diagnosis of the disease.

BACKGROUND OF THE INVENTION

Pancreas Disease (PD) is a serious disease affecting Atlantic salmon(Salmon salar L). The disease causes lesions in the pancreas, includingloss of pancreatic exocrine tissue and fibrosis, cardiac and skeletalmuscle myopathies. It is believed that other salmonoid species, such asrainbow trout, wild Atlantic salmon, could also be infected by PD.

Outbreaks of PD were first described in 1984 by Munro et al, inHelgoland Meeresuntersuchungen 37:571-586 (1984), but PD was recognisedas early as 1976. PD has also been reported in all of the major salmonfarming countries of the world, including Norway, Ireland, France, Spainand Western U.S.A. (see Kent et al, Bull. Eur. Ass. Fish Path. 7:29-31(1987); Poppe et al, in Bull. Eur. Ass. Fish Path. 9(4):83-85 (1989);and Raynard et al in Proceedings of a European Commission Workshop,Scottish Office Aquaculture Report No 1, p2-4 (1992)).

PD is known to affect fish in their first year in salt water and tospread rapidly in farmed fish held in sea cages. Ferguson et al (inJournal of Fish Diseases 9:95-98 (1986)) reported that affected fishwere thin, anorexic and lethargic with a tendency to congregate in cagecorners and to fail to maintain a horizontal position. In addition tothe primary pancreatic lesions, Ferguson et al supra reported that fishaffected by PD exhibited severe degenerative cardiomyopathy. Theseobservations were confirmed in a later study by Murphy et al (seeJournal of Fish Disease 15:401-408 (1992)) who found that cardiac andskeletal myopathy is exacerbated in fish infected with PD.

In Ireland over the period 1988-1992 PD resulted in 15-20% of recordedmortalities in salmon smolts in their first year at sea. The estimatedcost to the Irish industry in terms of loss of production is currentlythought to be around .English Pound.25 million per year. The current1994 production figures for Norway, Scotland and Ireland are as follows:

    ______________________________________               Tonnes of salmon                           Numbers of smolts put    Country    produced    to sea    ______________________________________    Norway     200,000     80 million    Scotland   55,000      20 million    Ireland    44,000       7 million    ______________________________________

McVicar et al postulated that PD was caused by an infectious agent. Thisproposition is supported by the results of epidemiological studies andtransmission experiments by various workers, which suggest an infectiousaetiology for the disease, (see McVicar in Aquaculture 67:71-78 (1987);McVicar in Bull. Eur. Ass. Fish Path. 10:84-87 (1990); Raynard et al,Dis. Aquat. Org. 15:123-128 (1993)); and Murphy et al (1992) supra).Recently Houghton (1994) 18: 109-118 reported that fish become resistantto re-infection after inoculation with PD, supporting the notion that PDis caused by an infectious agent. However, to date no infectious agenthas been isolated despite numerous attempts to do so (see McVicar (1987)supra; Munro supra; and Murphy supra).

SUMMARY OF THE INVENTION

The present invention reports the isolation of the causative agent of PDfor the first time. The causative agent has now been found to be aspherical virus of 65.5±4.3 nm in size, also referred to herein as FPDVvirus. Without projections it has a diameter of 46.8±2.5 nm, ischloroform and pH sensitive, resistant to inhibition by BUDR and onexamination by electron microscope morphologically possessessimilarities to a member of the Togavirus group. The Togavirus family iscomprised of 3 genera, Alphaviruses (27 species), Rubiviruses (1species) and Arteriviruses (1 species). When inoculated into freshwatersalmon parr and marine salmon post-smolts it produces pancreas diseasewith its associated morphological changes in the pancreas, heart, andskeletal muscle.

Viewed from one aspect, the present invention provides Fish PancreasDisease Virus (FPDV).

FPDV is a toga-like virus. It consists of spherical enveloped particles,having a particle size of 64-66 nm as measured by electron microscopyand a density of 1.2 g/ml in caesium chloride. Inoculation of 10³.5TCID₅₀ intraperitoneally into Atlantic salmon post-smolts held in seawater at 14° C. causes the fish to develop symptoms of pancreaticdisease, that is to become inappetant and to develop pancreatic acinarcell necrosis, cardiac necrosis and skeletal myopathy.

By FPDV we mean a virus having the above characteristics. The inventionis not limited to any particular virus strain of FPDV, howeverembodiments of the invention are directed to the specific strain(s) ofFPDV isolated and closely related strains thereof. By "closely relatedstrains" we mean any strain which shares similar genotypic and/orphenotypic characteristics to the strain(s) isolated. In particular thisphrase encompasses slightly modified forms of the virus which retainsubstantially the same functional activities. Thus, for example someamino acid or nucleotide additions, deletions or alterations have verylittle effect; if any, on the functional activities of the virus.

In particular, with FPDV we mean a fish virus which serologically reactswith convalescent anti-FPDV antiserum or antiserum raised against thedeposited FPDV sample (ECACC No. V94090731). More in particular a FPDVis a fish virus which gives a positive reaction with either of theseantisera in an indirect fluorescent antibody test (IFA).

Desirably the virus of the present invention is in a form substantiallyfree of other types of viral or microbial material.

A sample of FPDV has been deposited at European Collection of AnimalCell Cultures, Porton Down, Wiltshire, United Kingdom (ECACC) underDeposit No V94090731 on 7th Sep. 1994.

Further, the present invention provides polypeptides derived from FPDV(which term includes functional equivalents or parts of suchpolypeptides). The term "polypeptide" as used herein is not limitingwith regard to the size of the molecule and includes distinctive shortpeptides as well as large proteins.

The polypeptides of the present invention may be produced by anyconvenient method. For example, the polypeptides may be produced byharvest from active or attenuated forms of FPDV, including proteolytictreatment of such forms of FPDV. Suitable proteolytic agents arewell-known to those skilled in the art, and include enzymes such astrypsin or pepsin and chemical reagents such as sulphuric orhydrochloric acids. It is also possible to use detergents to solubilisevirus preparations to produce whole proteins that may be active.Alternatively, the polypeptides of the present invention may be producedby genetic engineering techniques. For example, an appropriateprotein-encoding portion of a nucleotide sequence may be expressed toproduce the required polypeptide. The required genetic engineeringtechniques are well-known to those skilled in the art, but in summary acDNA copy of at least the appropriate portion of the FPDV RNA genome isprepared. Suitable primers for cDNA production may include an oligo Tprimer, a primer designed from nucleotide information of a related virusor primers which are produced with random sequences. The DNA may then beplaced into an appropriate vector and optionally the proteins encodedthereby may be expressed by a compatible host. Optional steps includeinsertion of a suitable expression control sequence, clonal expansion ofthe recombinant vector and selection of the required recombinantconstruct.

As a general reference to genetic engineering techniques, mention may bemade of Maniatis et al, in Molecular Cloning a Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982.

The polypeptides of the present invention include all polypeptides ofFPDV (including functional equivalents or parts thereof) and thuscomprises polypeptides having a structural or a non-structural role inthe virus particle. With regard to the structural polypeptides of FPDVmention may be made of the core and envelope polypeptides of FPDV. Theinvention also covers a polypeptide comprising a surface epitope ofFPDV. The present invention also covers non-glycosylated andglycosylated forms of the polypeptides.

Viewed from a further aspect, the present invention provides a geneticconstruct comprising a nucleotide sequence derived from at least part ofthe genome of FPDV.

Thus the present invention provides a polynucleotide having a nucleotidesequence at least part of which corresponds to a nucleotide sequencederived from at least part of the genome of FPDV, which may include aprotein encoding region.

The phrase "derived" from includes identical and complementary copies ofat least a part of the genome of FPDV, whether of RNA or DNA and whetherin single or double-stranded form. The phrase "derived from" furtherincludes sequences with alterations which (due to the degeneracy of thegenetic code) do not affect the amino acid sequence of the polypeptideexpressed, as well as sequences modified by deletions, additions orreplacements of nucleotide(s) which cause no substantial deleteriouseffect to function (including the function of the polypeptideexpressed).

In particular, the genetic constructs of the present invention encompassthe naturally occurring genome of FPDV and cDNA equivalents thereof.Further, the genetic construct of the present invention includes allrecombinant constructs comprising nucleotide sequences derived from atleast part of the genome of FPDV. Such recombinant constructs may bedesigned to express only a particular polypeptide or polypeptides ofFPDV and may include non-FPDV (foreign) expression control sequences.Alternatively, the recombinant constructs may include an expressioncontrol sequence of FPDV, and optionally a non-FPDV (foreign) proteinencoding sequence.

In a particular embodiment, the present invention includes a vector(such as a cloning or expression vector) which comprises a geneticconstruct as defined above. Vectors include conventional cloning andexpression plasmids for bacterial and yeast host cells as well aseukaryotic virus vectors such as vaccinia, which may be useful forexpression of FPDV proteins in eukaryotic cell lines. Such a vector maybe used to transform a suitable host cell (either for cloning orexpression purposes) and the transformed host cell also forms a furtheraspect of the present invention. Suitable host cell types fortransformation with FPDV itself include Chinook salmon embryo (CHSE-214)cells, Atlantic salmon cell lines and Rainbow trout cell lines. However,if the vector produced is comprised only in part of a nucleotidesequence derived from FPDV it may be more appropriate to select a hostcell type which is compatible with the vector. Mention may be made ofprokaryotic host cells such as E coli and Yersinia ruckeri which havebeen used successfully for the expression of viral haemorrhagicsepticaemia rhabdovirus as well as eukaryotic host cells, includingyeasts, algae and fish, insect or mammalian cells in culture. Insectcells may be especially useful where a baculovirus expression system isused. Suitable host cells will be known to those skilled in the art.

In particular, the vector of the present invention may be based upon agenetically engineered version of the FPDV genome, which includes acoding sequence of a non-FPDV polypeptide and is able to express saidnon-FPDV polypeptide.

The genetic constructs, vectors and transformed host cells may be usedto express polypeptides, especially FPDV polypeptides.

The non-FDPV polypeptide may be, for example, a polypeptide from a fishdisease causative agent. The vector may thus be useful as a vaccine, theexpression of the non-FPDV polypeptide in vector-infected fish inducingan immune response to the fish disease causative agent.

There is evidence that fish acquire a strong immunity to PD after fieldand experimental exposure (see Raynard et al, Dis Aquat Org 15: 123-128(1993)). The isolation of FPDV will enable the development of antigenand nucleic acid detection systems which would aid in the rapiddiagnosis of PD and assist in more thorough investigation of thepathogenesis and epidemiology of this important fish disease. Thegenetic constructs and polypeptides of the present invention maytherefore be useful to produce a vaccine and/or diagnostic materialsagainst FPDV.

In a yet further aspect, the present invention provides a vaccine to PD,said vaccine comprising FPDV or a polypeptide derived from FPDV(including functional equivalents and parts thereof). In particular FPDVcould be used as a vaccine vector, that is could be geneticallyengineered as an expression vector having particular utility in vaccineproduction.

Thus the vaccine may be, for example, an attenuated or inactivated formof FPDV itself. Inactivated forms of FPDV may be produced by heating asample of FPDV, for example heating above 50° C., by treatment withchloroform, adjustment of pH or by any other suitable means. Attenuatedforms of FPDV may be produced by prolonged passage of the virus in cellculture, often of a different species, or by growth at progressivelyhigher temperatures, to select populations better adapted to a highertemperature. Development of plaque purification methods to selectvariants by plaque size has been used in other viruses and may besuitable here. Alternatively the vaccine may comprise a polypeptide ofFPDV, preferably a polypeptide which is immunogenic in fish (especiallyAtlantic salmon), that is to say the polypeptide induces an immunereaction in the fish. Such a polypeptide may be produced by anyconvenient means, for example by using genetic engineering techniques.

Vaccines to other togaviruses are known in the art and thus thetechniques of producing a suitable vaccine are available to the skilledpractitioner. Mention may be made to Roerig et al in High TechnologyRoute to Virus Vaccines, ed Driesman, Bronson & Kennedy 1985, Page 142and also to Leong et al in Annual Review of Fish Diseases (1993) pages225-240.

A suitable FPDV vaccine or non-FPDV vaccine, using FPDV as an expressionvector as described above, could be produced in a manner analogous toSemliki forest virus (SFV). With SFV, the production of a full-lengthcDNA clone, from which infectious RNA could be transcribed, and theelucidation of SFV molecular biology has facilitated the separation ofcDNAs that code for the RNA replication proteins from cDNAs coding forthe capsid proteins. A subgenomic mRNA encoding the capsid proteins canbe isolated from infected cells. This separation has been exploited toproduce "non-replicating" SFV particles comprising the normal viruscapsid enclosing an RNA that encodes the RNA replication proteins only.These particles are produced by co-transfecting cells with 2 differentRNAs each synthesised by in vitro transcription from distinct cDNAs.Transcript 1 encodes the proteins responsible for RNA replication andtranscript 2 codes for the proteins constituting the capsid andenvelope. Inside the transfected cell, both RNAs are replicated andtranslated. Due to its possession of the packaging signal, only the RNAencoding the replication proteins is encapsidated. The incorporation offoreign genes into the cDNA encoding the RNA replication capability hasallowed SFV to be exploited as a very efficient expression system. Thus,cells transfected with a modified transcript 1 along will expressforeign proteins. The potential of SFV as a vector vaccine is realisedwhen cells are co-transfected with the modified transcript 1 andtranscript 2. The outcome in this case is the production of"non-replicating" SFV particles which will infect cells and effectivelyproduce foreign proteins capable of invoking a protective immuneresponse.

Optionally the vaccine would be administered to young fish, for examplesalmon in the fresh-water stage. The vaccine may be added directly tothe water containing the fish. Alternatively, the fish (or a sample ofthe fish) could be inoculated directly. Where only a sample of the fishare inoculated, immunity may be conferred on the other fish due to thecontact with the vaccinated fish.

In another aspect, the present invention provides a diagnostic reagentfor PD, said reagent comprising a moiety capable of binding selectivelyto FPDV or to a component thereof.

Examples of said moiety include antibodies or other proteins able tobind selectively to FPDV itself or to a polypeptide thereof, lectinswhich bind selectively to FPDV, oligosacharides or glycosylatedpolypeptides thereof, and polynucleotides having sequences which arecomplementary to at least a portion of the FPDV genome.

Optionally the diagnostic reagent may include a marker, such as aradioactive label, a chromophore, fluorophore, heavy metal, enzymiclabel, antibody label or the like. Optionally, the diagnostic reagentmay be immobilised (for example on a bead, rod, vessel surface ormembrane) and the sample to be tested is brought into direct contactwith said diagnostic reagent.

Antibodies specific to FPDV which may be utilised as said moiety in thediagnostic reagent form a further aspect of the present invention Ifrequired the antibodies may be monoclonal antibodies.

In a yet further aspect, the present invention provides a method ofisolating FPDV. This method comprises identifying fish suffering fromPD, preferably fish in the acute stage of PD (as defined by Munro et al,supra). Affected tissues (such as the pancreas or kidney) areco-cultivated with Chinook salmon embryo (CHSE-214) cells for anappropriate length of time, for example up to 35 days, especiallyapproximately 28 days. The co-cultivated cells are then passaged throughCHSE cells.

The present invention also provides a method of diagnosing PD, saidmethod comprising the following steps):

i) contacting a test sample with a diagnostic reagent of the presentinvention to produce a reagent complex;

ii) optional washing step; and

iii) determining the presence, and optionally the concentration, ofreagent complex and thus the presence or amount of FPDV in the sample.

The method of diagnosis may be performed on any sample suspected tocontain FPDV. For example, tissue samples (for example kidney, spleen,heart, pancreas, liver, gut or blood) of the fish may be subjected tothe diagnosis procedure. Generally, it is preferred for a blood sampleto be tested, thus providing a non-fatal diagnosis. It may also bepossible for the diagnostic test to be performed on a sample of thewater which has been used to contain the fish.

The present invention also provides a method of producing FPDV and amethod of producing a polypeptide derived from FPDV.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures of the Application may be briefly discussed as follows:

FIG. 1a: Uninfected CHSE-214 cells (Magnification×750);

FIG. 1b: CHSE-214 cells, 8 days post-inoculation with FPDV(Magnification×750);

FIG. 2. Growth of SPDV in CHSE-214 cells.

FIG. 3: Transmission electron micrographs of glutaraldehyde fixed, FPDVinfected CHSE-214 cell culture fluid. Most of the virus particles havesurface projections, but little internal structure detail

Bar=100 nm;

FIG. 4: Significant pancreatic acinar cell loss, typical of pancreaticlesions induced by FPDV at post-inoculation day 21

Bar=50 μm;

FIG. 5: Multifocal cardiomyocytic necrosis which occurred concurrentlywith the pancreatic lesions at post-inoculation day 21 (.sub.→)

Bar=20 μm;

FIG. 6: Degeneration of aerobic (red) skeletal muscle showing increasedendomysial connective tissue, proliferation of sarcolemmal cells andhyaline degeneration of muscle fibres at post-inoculation day 21

Bar=50 μm; and

FIG. 7: Hyaline degeneration of anaerobic (white) skeletal muscle fibresshowing centralisation of muscle fibre nuclei and phagocytosis of fibrecontents

Bar=50 μm.

FIG. 8: Virus Isolate×100,000

The present invention will now be further described with reference tothe following, non-limiting, examples.

EXAMPLE 1 Isolation and Cell Culture of Virus

Cell Cultures

For virus isolation the chinook salmon embryonic cell line (CHSE-214,Nims et al, 1970) was used throughout the investigation. Other celllines used include epithelioma papulosum cyprini (EPC), fathead minnow(FHM), bluegill leponis macrochirus (BF2), Atlantic salmon (AS), rainbowtrout gonad (RTG-2) and rainbow trout fibroblast (RTF) cells. Cells weremaintained in Eagle's minimum essential medium (MEM) containing Earle'ssalts and sodium hydrogen carbonate 2.2 g/l supplemented with 200 mML-glutamine, 1% non-essential amino acids, 0.01M Hepes, penicillin 100IU./ml, streptomycin 100 μg/ml and 10% foetal bovine serum (FBS) Gibco,Scotland. Cells were propagated in either 150 cm² flasks or 24 wellplates (Costar 3524) at 20° C.

Plates were incubated in closed containers in 3% CO₂ /97% airatmosphere. For maintenance of cells during virus isolation, amaintenance medium (MEMM) was used comprised of MEM with antibioticsincreased as follows; penicillin 500 IU ml⁻¹, streptomycin sulphate 500μg ml⁻¹, amphotericin B 0.625 μg ml⁻¹ ; and FBS was reduced to 2%.

Original Virus Isolation

Samples of kidney, spleen, heart, liver, pancreas and gut were takenfrom 20 individual fish during the acute phase of an outbreak ofPancreas disease in farmed Atlantic salmon on the west coast of Ireland.Samples from each fish were treated separately.

Co-cultivation

For isolation attempts by co-cultivation, half portion aliquots of eachkidney were fragmented by placing them in a 2 ml syringe and forcingthem through a 16 gauge hypodermic needle into 10 ml of maintenancemedium (MEMM). The suspension of tissue pieces obtained were inoculatedinto monolayers of CHSE-214 cells prepared 24 hours previously. One mlof the tissue suspension was inoculated into each well of a 24 wellplate and incubated at 15° C. for 28 days or until a cytopathic effect(CPE) was observed, when passage into CHSE cells, without freezing andthawing, and incubation for a further 28 days was carried out.

Tissue Homogenates

The remaining kidney portions and other tissues from each fish werepooled and 10% homogenates prepared with MEMM using a pestle and mortar.These were centrifuged at 2500 g for 15 minutes and 0.1 ml of thesupernatants were inoculated at final dilutions of 1:20, 1:50 and 1:100in MEMM into 24 well plates containing CHSE-214 cells. These wereincubated at 15° C. for up to 28 days or until the appearance of CPE,when passage into CHSE cells and incubation at 15° C. to a further 28days was carried out.

Viral Growth Curve in CHSE-214 Cells

Virus growth in CHSE-214 cells was measured by inoculation of 0.1 ml ofFPDV on to CHSE-214 cells in 24 well plates at a multiplicity ofinfection (MOI) of 1 TCID₅₀ /cell and allowing virus to absorb for 1hour at 15° C. The innoculum was removed and the cells washed threetimes with MEMM, before replacing with 1 ml MEMM. Samples were removedfor assay on postinoculation days (PID) 0, 2, 4, 6, 8, 10, 12, 14, 21and 28 as follows. For extra-cellular virus samples, half of the culturemedium was removed from each of 4 wells, pooled and centrifuged at 800×gfor 5 minutes to remove the cells. The supernatant containedextra-cellular virus. For total virus samples, the remaining 0.5 ml ofculture medium, along with the adherent cells removed by scraping, werepooled, and frozen and thawed once. Both samples were assayed separatelyfor virus infectivity by titration in CHSE-214 cells incubated at 15° C.for 14 days. The 50% end points in this and all subsequent tests wereestimated by the method of Reed and Muench (1938) Am. J. of Hygiene 27:493-497.

Growth of the virus in cell cultures

Examinations for cytopathic effects of FPDV in epithelioma papillomacyprini (EPC), fathead minnow (FHM), bluegill leponis macrochirius(BF2), Atlantic salmon (AS) (Flow, Scotland) rainbow trout gonad (RTG)cells and a rainbow trout fibroblast cell (RTF) line produced in thislaboratory were carried out. Cultures were inoculated with ten-folddilutions of a virus pool containing 10⁷ TCID₅₀ ml⁻¹, incubated at 15°C. and examined for evidence of CPE over 14 days. Any CPE was noted andcultures were tested for virus growth by titration of culture fluids inCHSE-214 cells. All cultures were given one further passage for 14 daysin the same cells at 15° C., and checked again by virus growth inCHSE-214 cells before discarding.

RESULTS

Virus Isolation

Virus was isolated from two out of twenty kidney tissues submitted forexamination. These had been co-cultivated with CHSE-214 cells for 28days and then given further passages in CHSE-214 cells. No CPE was seenin the original co-cultivation cultures. On passage however, smalldiscrete groups of cells which were pyknotic, vacuolated and irregularin appearance could be observed after 10 days incubation. After fourfurther passages in CHSE-214 cells the CPE had become widespread (seeFIG. 1). Virus titres of 10⁸.5 TCID₅₀ /ml were routinely obtained. Mostof the affected cells remained attached to the monolayers. No syncytiaor inclusion bodies were observed in any of the cultures. No virus wasisolated from any of the tissue homogenates inoculated.

Description of Cytopathic Effects in CHSE-214 Cells

In the early passages small discrete groups of cells became pyknotic,vacuolated and irregular in appearance. These increased over three weeksuntil up to the three-quarters of the monolayer became affected.Infected cells did not detach from the surface of the culture plate.When the virus became cell adapted the CPE appeared as early as 4 daysand were usually complete by 14 days.

Growth in Various Call Cultures

Growth of the virus in CHSE-214 cells is shown in FIG. 2. Highest totalvirus levels were achieved by 6-8 days post-inoculation, whereasextra-cellular virus peaked around 11 days post-inoculation and remainedhigh for up to 14 days post-inoculation. No CPE were observed in AS,BF2, FHM, EPC, or RTG-2 cells and there was no evidence of growth ofFPDV in these cells. However, in the RTF cell line although no CPE wereobserved, FPDV titres reached 10⁶ TCID₅₀ ml⁻¹ on both first and secondpassage in these cells.

EXAMPLE 2

Chloroform Sensitivity

Sensitivity to chloroform was determined by adding 0.05 ml chloroform to1 ml of virus. The mixture was shaken for 10 minutes at ambienttemperature then centrifuged at 400×g for 5 minutes to remove thechloroform. Residual infections virus was detected by titration inCHSE-214 cells. A control consisted of 0.05 ml of MEMM added to 1 ml ofvirus instead of chloroform. Infectious Pancreatic Necrosis Virus (IPNV)and Viral Haemorrhagic Septicaemia virus (VHS) were also included asnegative (non-sensitive) and positive (sensitive) virus controlsrespectively.

RESULTS

The infectivity of the isolate and VHS virus was reduced followingexposure to chloroform indicating the presence of an envelope containingessential lipids.

                  TABLE 1    ______________________________________    Sensitivity of FPDV to chloroform    Virus concentration Log TCID.sub.50 /ml    Virus          Control Treated    ______________________________________    FPDV           7.5     <1.0    IPNV           7.5     7.5    IHN            6.0     <1.0    ______________________________________

In contrast IPNV infectivity was not affected when treated in the sameway.

EXAMPLE 3

Stability at pH 3.0

Stability at pH 3.0 was determined by adding 0.1 ml virus to 0.9 ml MEMadjusted to pH 3.0, holding for 4 hours at 4° C. then checking forresidual infections virus by titrating in CHSE-214 cells at 15° C. for14 days. The experiment was repeated with FPDV added to MEM at pH 7.2 asa control. IPNV was included as a pH 3.0 stable virus control andinfectious hematopoietic necrosis virus (IHN) was used as a pH 3.0sensitive control

RESULTS

                  TABLE 2    ______________________________________    Sensitivity of FPDV to pH 3.0    Virus concentration Log TCID.sub.50 /ml    VIRUS          pH 3.0  pH 7.2    ______________________________________    FPDV           <1.0    6.5    IPNV           7.5     7.5    IHN            <1.0    6.0    ______________________________________

The infectivity of the isolate was lost when it was exposed to pH 3.0.IPNV was not affected.

EXAMPLE 4

Temperature Stability

Aliquots of viral suspension were heated for 30 minutes at 15, 25, 37,45, 50, 55 or 60° C. and then cooled immediately by immersion in icedwater. The concentration of infectious virus remaining was assayed bytitration in CHSE-214 cells incubated at 15° C. for 14 days.

RESULTS

The infectivity was not affected at 4, 15, 25° C. but was reduced at 37and 45° C. No infectious virus was detected after 30 minutes at 50° C.

Table 3

Stability of FPDV held at different temperatures for 30 minutes.Residual virus assayed for infectivity in CHSE-214 cells, incubated at15° C. for 14 days.

    ______________________________________    Temp (° C.)               Virus concentration Log TCID.sub.50 /ml    ______________________________________    4          7.5    15         7.5    25         7.5    37         6.5    45         5.5    50         --    ______________________________________

EXAMPLE 5

Haemagglutination

Tests for haemagglutination were carried out with chicken, guinea pig,rainbow trout, and Atlantic salmon erythrocytes is U-bottomed 96 wellplates, by adding 0.1 ml of a 0.8% suspension of red blood cells inphosphate buffered saline (PBS) pH 7.2 to 0.1 ml of a FPDV pool preparedin CHSE-214 cells with a titre 10⁷ TCID₅₀ /ml and incubating at 4° C.,15° C., and 37° C. Tests were examined after 1, 3 and 18 hours.

RESULTS

No haemagglutination was observed at any of the temperatures, or withany of the eythrocytes selected.

EXAMPLE 6

Nucleic Acid Inhibition Test

The nucleic acid type of the virus was determined by growing the virusin the presence of the DNA inhibitor 5-bromo-2'-deoxyuridine (BUDR) withand without thymidine. Groups of 4 wells in each of three 24-well platescontaining CHSE-214 cells were inoculated with 0.1 ml of ten-folddilutions of virus, which were allowed to absorb for 1 hour at 15° C. Toeach plate 1 ml of MEMM alone, MEMM with 1 mM/ml BUDR or MEMM with 1mM/ml BUDR and 1 mM/ml thymidine, were added. The plates were incubatedat 15° C. for 14 days and examined for CPE. A fish RNA virus (IPN) and aDNA virus (lymphocystis) grown in BF2 cells were included as controls.

RESULTS

                  TABLE 4    ______________________________________    Replication of FPDV in CHSE-214 cells in the    presence of 1mM 5-bromo-2'-deoxyuridine    (BUDR)    Virus concentration Log TCID.sub.50 /ml    Virus    MEMM    MEMM + BUDR MEMM + BUDR + THY    ______________________________________    FPDV     7.0     7.25        7.0    IPNV     7.5     7.25        7.5    Lymphocystis             7.2     5.0         7.0    virus    IHN      6.0     nd          nd    ______________________________________     nd = Not done

The virus titre of the isolate and IPNV was not affected by the presenceof BUDR in the medium whereas the fish DNA virus was inhibited. Thisindicates that the genome of the isolate is comprised of RNA.

EXAMPLE 7

Negative Contrast EM Examination

Virus suspensions for EM examinations consisted of either FPDV infectedcell culture medium used without prior fixation or after the addition ofglutaraldehyde (2% final conc.) for 1 hour at 4° C. and subsequentultracentrifugation at 100 000×g for 4 hours, then resuspension of thepellet in a few drops of distilled water. A carbon coated copper gridwas placed on top of a drop of virus suspension and allowed to stand for10 minutes. Excess fluid was drained off and the grid was stained with2% phosphotungstic acid (PTA) (pH 7.2) for 1 minute. It was examined ina Hitachie H7000 transmission EM at ×50,000 magnification.

RESULTS

Virus preparations not fixed in glutaraldehyde before EM examinationcontained mostly disrupted particles. indicating that pre-fixation isrequired to preserve the intact virion.

EM examination of the glutaraldehyde fixed material revealed thepresence of circular particles measuring 65.5±4.3 nm (FIG. 3). Thesepossessed an inner core of indefinite structure and were surrounded byan outer fringe of what appeared to be club-like projections. Manypartially disrupted particles were also present. In the unfixedpreparations only a few complete particles were seen and this indicatesthat the free virion is fragile and easily disrupted during preparationfor EM examination.

EXAMPLE 8

Growth and Concentration of Virus

Growth of Virus in CHSE-214 Cells

Virus growth in CHSE-214 cells was measured by inoculation of 0.1 ml ofFPDV on to CHSE-214 cells in a 24 well plate at a multiplicity of 1.0TCID₅₀ /cell and allowing it to absorb for 1 hour at 15° C. The viruswas then removed and the cells washed twice with MEMM before replacingwith 1 ml MEMM. Samples were removed for assay on days 0, 7, 11, 14, 21,28 post inoculation as follows. Half of the culture medium was removedfrom each of 4 wells pooled and centrifuged at 800×g for 5 minutes toremove the cells. The remaining 0.5 ml of culture medium along with theadherent cells, removed by scraping were pooled, and then frozen andthawed once. Both samples were assayed separately for virus infectivityby titration in CHSE-214 cells at 15° C. for 14 days.

Caesium Chloride Gradient Centrifugation

FPDV was inoculated into CHSE-214 cells at an MOI of 1. Cells and medium(400 ml) were harvested at 8 days post inoculation and frozen and thawedonce at -70° C. before centrifugation at 10,000×g for 30 minutes in aBeckman Type 35 angle rotor to remove cell debris. The supernatant wassubjected to ultracentrifugation of 100,000×g for 4 hours. The resultantpellet was resuspended in a total of 2 ml of PBS pH 7.2 and layered overa discontinuous CsCl gradient comprised of 5 ml of 1.3 g/ml CsCl and 4.5ml of 1.22 g/ml CsCl. This was centrifuged at 100,000×g for 19 hours at4° C. 20 fractions were collected and tested for infectivity in CHSE-214cells incubated at 15° C. for 14 days. The density of the fractionscontaining infective virus was determined using a refractometer.

RESULTS

Infectivity was detected in fractions from CsCl gradients with densitiesfrom 1.08 to 1.26 g/ml. The maximum infectivity was observed at adensity of 1.2 g/ml and this fraction also contained the greatest numberof complete virus particles as assessed by EM examination.

EXAMPLE 9

Serological Tests

FPDV was tested for neutralisation by hyperimmune rabbit sera againstInfectious Haematopoietic virus (IHN), Viral Haemorrhagic Septicaemiavirus (VHS), Infectious Pancreatic Necrosis virus, strains Sp, Ab, andVR-299 (IPNV), Equine arteritis virus (EAV), Bovine Viral Diarrhoeavirus (BVD), and Rubella virus. Equal volumes of 200 TCID₅₀ /0.1 ml ofFPDV was added to 0.1 ml of two-fold dilutions of antisera and incubatedat 15° C. for 1 hour. The mixtures were then inoculated into CHSE-214cells in 24 well plates, 0.1 ml per well, allowed to absorb for 1 hour,1 ml MEMM added and incubated at 15° C. for 14 days, and the culturesexamined microscopically on alternate days for evidence of CPE. Titreswere calculated by the method of Reed and Meunch, 1938 in Amer. J. ofHygiene 27:493-497.

RESULTS

FPDV was not neutralised by antisera to IHNV, VHSV, IPNV, EAV, BVD orRubella, indicating that it was not related to these virus groups.

EXAMPLE 10 TRANSMISSION EXPERIMENT

1) Materials and Methods

a) Fresh-water Fish

Atlantic salmon parr of mean weight 20 g±2.9 g were maintained incircular 1.2 m diameter tanks containing liters of partiallyre-circulated spring source water at 10-12° C. The fish were kept inthese tanks for 2 weeks prior to inoculation to acclimatise and were fedon a commercially prepared diet to satiation. Tissues were removed from10 fish and examined for the presence of Infectious Pancreas Necrosisvirus (IPNV) and Pancreas Disease. Before inoculation fish wereanaesthetised with 3-aminobenzoic acid ethyl ester (MS222).

b) Marine Fish

Atlantic salmon post-smolts of mean weight 87 g were maintained in 2×1.5m tanks containing sea water in a flow through system at 12-15° C. Theywere kept for 2 weeks to acclimatise prior to inoculation, and samplesof tissue from 10 fish were cultured for the presence of IPNV andexamined histologically for evidence of Pancreas disease.

2) Experimental Procedures

Inoculum 1

A FPDV pool was prepared by inoculating virus into CHSE-214 cells at amultiplicity of infection of 1 TCID₅₀ per cell and harvesting after 8days incubation at 15° C. The cells were disrupted by freezing andthawing once at -70° C. and the cell debris was removed bycentrifugation at 10,000×g for 30 minutes. The resultant virus pool,titre 10⁷.0 TCID₅₀ /ml was filtered through a 0.22 micron porosityMillipore filter and 0.1 ml inoculated intraperitoneally into each of100 fish. Fifty fin-clipped un-inoculated fish were added to each of thetanks as in-contact fish.

Inoculum 2

As controls 100 fish were inoculated with a lysate from un-infectedCHSE-214 cells prepared in exactly the same manner as the virus infectedcells, and 50 additional in-contact control fish were added.

Sampling

On days 6 or 7, 10, 14 or 15, 21, 28, 35, 42 post-inoculation, samplesof heart, spleen, kidney, liver, caecae/pancreas and muscle were takenfrom 10 test and 10 control fish for histological examination. Heart,spleen, kidney and caecae/pancreas samples were also taken from the samefish for virus isolation until day 28. At days 14 or 15, 21, 28, 35 and42 post-inoculation 5 in-contact fish were removed and tissues sampledfor histological examination. In-contact fish were sampled for virusisolation on days 14 or 15 and 21 only.

Histology

Samples for histological examination were fixed in 10% formaldehyde inbuffered saline pH 7.0, embedded in paraffin wax and 5 micron sectionscut on a Reichert Ultracut S microtome. These were stained withhaematoxylin and eosin.

Virus isolation

Tissues were prepared as 10% homogenates in MEMM, using mortars andpestles, centrifuged at 2,500×g for 15 minutes then inoculated at finaldilutions of 1:20 and 1:10 into each of 2 wells in a 24 well platecontaining CHSE-214 cells and incubated at 15° C. for 28 days. Samplesshowing no CPE were given one further passage into CHSE-214 cells beforebeing considered negative.

RESULTS

Clinical and Pathological Lesions in Freshwater Salmon Parr

When the virus was inoculated into freshwater salmon parr, some of theparr stopped feeding and faecal casts were observed. Acute pancreaticacinar necrosis was detected from 6-10 days post inoculation (p.i.). Thenecrosis was focal to diffuse in distribution. Between day 14 and 21many of the fish had significant acinar loss but pockets of survivingacinar tissue were detected especially around the islets of Langerhansand larger intralobular ducts in some fish. There was mild fibrosis ofthe periacinar tissue. Concurrent multifocal myocytic necrosis anddegeneration was detected from day 6 p.i. in all fish with pancreaticlesions. In week 3 post-inoculation the hearts appeared hypercellularwith apparent proliferation of myocytic and sub-endocardial cells withmyocytic nuclear enlargement. Mild skeletal muscle lesions were detectedfrom 21 days p.i. In contact fish developed similar lesions after a 2week delay. No histological lesions were detected in the negativecontrols.

Clinical and Pathological Lesions in Seawater transmission experiment

By day 7, the fish inoculated with FPDV became anorexic and there was anincrease in faecal casts in the tank. Focal to severe diffuse acinarcell necrosis with concurrent multifocal cardiomyocytic necrosis wasconsistently observed from day 7. Skeletal muscle fibre degeneration wasdetected from day 15, affecting both red and white skeletal musclefibres. These muscle lesions increased in frequency and severity at days35 and 42. Typical lesions observed at day 21 are illustrated in FIGS. 4to 7. All the cohabitant fish developed similar lesions after a 2 weekdelay. No clinical signs or lesions were detected in any of the controlfish.

Isolation of Virus in Transmission Experiments

Virus was isolated from all tissues of the inoculated fish, but atdifferent times post-inoculation (Table 5), with the heart tissue givingthe best success rate.

                  TABLE 5    ______________________________________    Transmission Experiment - Fresh-water fish.    Virus isolations from 10% tissue homogenates    inoculated into CHSE-214 cells and incubated    at 15° C. for 14 days.    Days post-inoculation    Tissue   6          10    14      21  28    ______________________________________    kidney   +          +     +       +   -    spleen   -          +     +       -   -    heart    +          +     +       +   +    pancreas -          -     +       +   +    liver    -          +     +       -   -    gut      -          +     +       +   +    ______________________________________

In contact fish from the same tank became infected with the virus,although none of the control fish were found to contain virus. Theresults for the sea-water fish are given in Table 6. No IPN virus wasdetected at any stage in the transmission study.

                  TABLE 6    ______________________________________    Virus isolation in transmission experiment    (sea-water fish)            Number positive/number examined            Days post-inoculation    Fish      7         10     15     21   28    ______________________________________    Inoculated              0/10      0/10    0/10  0/10 0/10    controls    Virus     7/10      8/10   10/10  3/10 ND*    inoculated    Not    inoculated              ND*       ND*    5/5    5/5  ND*    (in-contacts)    ______________________________________     ND* = Not Done

Summaries of the histological results are shown on Table 7 and 8.

                  TABLE 7    ______________________________________    Summary of Histological Findings in    Experimental Transmission Studies Using FPDV    in Fresh-water Fish           Days post infection    Tissue   4        7      14       21   28    ______________________________________    Pancreas -        +      +        +    +                      7/10   10/10    10/10                                           2/10    Heart    -        +      +        +    +                      7/10   10/10     9/10                                           2/10    ______________________________________     No lesions detected in any negative controls

                  TABLE 8    ______________________________________    Summary of Histological Findings in    Experimental Transmission Studies using FPDV    in Seawater Salmon Smolts           Days post infection    Tissue   7         10     15      21   28    ______________________________________    Pancreas +         +      +             9/10      10/10  10/10   10/10                                           9/10    Heart    +         +      +             6/10      10/10  10/10   10/10                                           6/10    Muscle   0         0       2/10    8/10                                           9/10    ______________________________________     No lesions detected in any negative controls.

EXAMPLE 11 INACTIVATED FISH PANCREAS DISEASE VIRUS (FPDV) VACCINEExperimental Protocol

AIM OF STUDY

To investigate if inactivated FPDV with added adjuvant, when inoculatedinto salmon parr, can protect the fish from challenge with live FPDV.

Materials and Methods

Virus used in Preparation of the Vaccines

11th passage FPDV was grown in CHSE cells and harvested at 6 days postinoculation and centrifuged at 1000×g for 15 minutes 600 ml of theresulting supernatant was passed through an Amicon filter producing 25ml of concentrated FPDV at a titre of 10⁷.5 TCID₅₀ ml⁻¹.

Virus Inactivation Method

A. 10 ml FPDV as prepared above +0.2% beta-propiolactone (BPL)+2 dropsNaOH

B. 10 ml FPDV as prepared above +0.1% formalin (35-38%)

C. MEMM (maintenance medium).

Aliquots of A, B and C were stored at +4° C. for 24 hours beforeaddition of a suitable adjuvant. Adjuvant was added after virus wasinactivated and both virus and control were received and inoculated intofish 8 days after initial inactivation.

Vaccination Protocol

FISH:Salmon parr, average wt 27 g, were kept in freshwater flow-throughsystem for 1 or 2 weeks pre-vaccination. (Water temperature range 10-14°C.).

Blood and tissue samples were taken from 5 fish per group prior tovaccination.

0.1 ml of inactivated-adjuvanted FPDV as prepared in A and B and theadjuvant control group C (MEMM plus adjuvant) was inoculatedintraperitoneally (I/P) into 50 fish per group.

Sampling

10 fish from each group were sampled 1 week before challenging withFPDV.

Bloods and histopathological samples were taken and checked for evidenceof pancreas disease (PD).

No evidence of PD was found histologically or by virus isolation.

Challenge Protocol

0.1 ml of live 10⁴.5 TCID₅₀ ml⁻¹ of 9th passage FPDV was inoculated I/Pinto each fish in the 3 test groups at 28 days post-vaccination.

A fourth group D of 50 naive unvaccinated fish, were also inoculated I/Pwith the same amount of FPDV to act as a positive control.

After 10 and 14 days post challenge, 5 fish were sampled from eachgroup.

Blood samples for antibody tests, kidney and heart tissue samples forvirus isolation, and pyloric caeca, heart and muscle samples forhistology were taken.

    ______________________________________    VACCINE EXPERIMENT 1    10 DAYS        14 DAYS      21 DAYS    Group/          Histology        Histology    Histology    No    Panc   Heart  Virus                             Panc Heart                                       Virus                                            Panc Heart                                                      Virus    ______________________________________    A1    +      +      +    +    +    ND    A2    +      +           +    +    A3    +      -           +    +    A4    +      -           +    +    A5    +      -           +    +    B1    -      -      -    -    -    -    -    -    -    B2    -      -           -    -         -    -    B3    -      -           -    -         -    -    B4    -      -           -    -    B5    -      -           -    -    C1    -      -      +    +    +    ND    C2    +      +           +    +    C3    +      -           +    +    C4    +      +           -    -    C5    +      +           +    +    D1    +      +      +    +    +    +    D2    +      +           +    +    D3    +      +           +    +    D4    +      +           +    +    D5    +      +           +    +    ______________________________________     1 + = lesions or virus present     2 - = no lesions or virus present     3 ND = not done     4 GROUP A = FPDV + 0.2% BPL + ADJUVANT     5 GROUP B = FPDV + 0.1% FORMALIN + ADJUVANT     6 GROUP C = CONTROL MEMM + ADJUVANT     7 GROUP D = UNVACCINATED CONTROLS

EXAMPLE 12 INDIRECT FLUORESCENT ANTIBODY TEST (IFA) FOR DETECTION OFFPDV ANTIGEN IN CELL CULTURES

Introduction

The test involves addition of serum, containing FPDV antibody, to CHSEcells suspected of being infected with the virus. If virus is presentthe antibody will bind to the antigen present. Anti-salmon IgM preparedin rabbits is then added and will attach to the bound antibody. Thisattachment is detected by the addition of anti-rabbit serum conjugatedto fluorescein isothiocyanate (FITC) which can be viewed as greenfluorescence using a fluorescent microscope.

Materials

CHSE-214 cells are grown on 11 mm diameter coverslips in 24-well plates(Costar) by seeding 45,000 cells in 0.5 ml of growth medium (MEM+10%foetal bovine serum) per coverslip. The cultures are incubated at 20° C.in a closed container with 3% CO₂ /air mixture for 48 hours before use.

Positive serum--convalescent serum from FPDV affected salmon used at1/50 dilution in phosphate buffered saline (PBS) pH 7.2.

Negative control serum--salmon serum with no antibody to FPDV diluted1/50 in PBS.

Rabbit anti-salmon IgM--obtained commercially from Soren Schierbeck &Co, Helsingor, Denmark, diluted 1/50 in PBS.

Anti-rabbit Ig/FITC--obtained commercially from Nordic Imm. Labs.,Berks, England, diluted 1/1000 in PBS.

Method

Material suspect of containing FPDV is added to the coverslips in wellscontaining 1 ml MEMM (maintenance medium) and incubated for 5 days at15°0 C. in a closed container with 3% CO₂ /air mixture. The coverslipsare washed twice in PBS and fixed in acetone for 5 minutes then storedat +4° C. until used. Negative control cultures, without suspectsamples, as well as positive control cultures, containing FPDV, areprepared in the same way.

Test and control coverslips are overlaid with 50 μL of positive andnegative sera and incubated at 35° C. for 30 minutes in a humidified 100mm square petri dish. PBS is added to separate coverslips as reagentcontrols. All coverslips are then rinsed in PBS for 6 minutes (3 changesof 2 minutes each) and excess PBS is drained off on tissue paper. 50 μLof anti-salmon IgM is added to all coverslips and incubated for 30minutes at 35°0 C. Further rinsing is carried out in PBS for 6 minutesand 50 μL of anti-rabbit/FITC is added to all coverslips which areincubated for 30 minutes at 35°0 C. A final rinsing is carried out for 6minutes in PBS and the coverslips are examined under a 40× objective ona Labophot 2 fluorescence microscope with the coverslips under bufferedglycerol saline pH 9.2. Green fluorescence similar to the positivecontrols, with absence of fluorescence in the negative controlsindicates presence of FPDV antigen in cultures.

EXAMPLE 13 INDIRECT FLUORESCENT ANTIBODY TEST (IFA) FOR DETECTION OFANTIBODY TO FPDV IN SALMON SERUM

Introduction

The test involves addition of salmon serum to FPDV infected CHSE-214cells. If antibody to FPDV is present in the serum it will bind to thevirus infected cells. Anti-salmon IgM prepared in rabbits is then addedto the preparation and will attach to any bound antibody. Thisattachment is recognised by addition of anti-rabbit serum conjugated tofluorescein thiocyanate (FITC) which can be viewed as green fluorescence(FITC) under a fluorescence microscope.

Materials

FPDV infected cells are prepared by adding virus to CHSE-214 cellsuspension containing 200,000 cells per ml, at a multiplicity of 1 andseeding 40 μL into each well of 12 well multispot slide (Hendley,Essex). The cultures are incubated in a 3% CO₂ /air atmosphere in aclosed container at 20° C. for 24 hours. They are then transferred to a15° C. incubator and incubated for a further 3 days before rinsing twicewith phosphate buffered saline (PBS) pH 7.2, fixing with acetone for 5minutes and storing at +4° C. until used. Control CHSE-214 cultureswithout virus are prepared in the same way.

Test sera--salmon serum for antibody testing.

Positive control serum--salmon serum shown to have neutralising antibodyby serum neutralising antibody test (SNT).

Negative control serum--salmon serum with no antibody by SNT.

Rabbit anti-salmon IgM--obtained commercially from Soren Shierbeck & Co,Helsingor, Denmark.

Anti-rabbit Ig/FITC--obtained commercially from Nordic Imm. Labs. Berks,England.

Method

FPDV positive and negative multispot wells are overlaid with 30 μL ofsalmon test sera diluted 1/20 with PBS pH 7.2. Positive and negativesera at 1/20 dilution in PBS are also included in separate wells. Twowells on each slide also have PBS added as controls and slides areincubated at 35° C. in a humidified 100 mm² petri dish for 30 minutes.Following rinsing in PBS (three rinses of 2 minutes each) 30 μL of antisalmon IgM at a 1/50 dilution in PBS is added to all wells and theslides incubated for a further 30 minutes at 35° C. After rinsing for 6minutes in PBS, 30 μL anti-rabbit IgM at a 1/100 dilution in PBS isadded to each well and the slides incubated for 30 minutes at 35° C. Afinal rinse in PBS for 6 minutes is carried out and the presence offluorescence detected using a 40× objective and a Labophot 2 fluorescentmicroscope with the slides under buffered glycerol saline pH 9.2. Adistinctive green fluorescence in test and positive serum wells and notin the controls indicates the presence of antibody to FPDV in thesesera. Antibody titrations are carried out using the same method.

EXAMPLE 14

Virus RNA: ¹⁴ C Uridine

Although less commonly use than tritium, this label was chosen becauseit is detectable using X-ray film.

Labelled, extracelluar virus was pelleted at high speed and extractedfor analysis by formaldehyde-gel electrophoresis. In the firstexperiment, in which Act D was present during labelling period (day 2 to5), no label was detected on the gel.

RNA present in Act D-treated, infected cells was also investigated.Following extraction by the NP40 lysis method a smear of labelled RNA,increasing in intensity as molecular weight decreased, was observed. Nodistinctive candidate viral RNAs could be detected.

Larger volumes of extracelluar virus, labelled with ¹⁴ C and ³ H-uridinein the absence of Act D, were pelleted and extracted with the Gensys RNAisolator method. On a formaldehyde gel showing very good resolution oflabelled ribosomal RNAs (from infected cells), high-speed pellet virusRNA migrated as a smear. Most of the RNA was of low molecular weight,corresponding in mobility to tRNA or slightly greater, but the smearextended in size to greater than the 5000nt (larger rRNA species). Longexposures of this gel indicated the presence of 2 faint bands, onecorresponding in size to the 28S rRNA and the other at a positioncorresponding to about 10-15 kb. The larger species possesses a mobilitysimilar to that expected for togavirus/flavivirus. One possibleexplanation for the smaller RNA is that we have detected the subgenomicRNA, produced by togaviruses.

EXAMPLE 15 FISH PACREAS DISEASE VIRUS (FPDV) TRANSFECTION RESULTS

Transfections were successfully performed with (a) RNA extracted frominfected CHSE cells (Intracellular RNA), and (b) RNA extracted fromvirus present in the tissue culture supernatant (Extracellular virusRNA).

RNA Preparation

Intracellular RNA

Monolayers of CHSE cells in 75 cm² plastic flasks were infected withFPDV (typically 10⁴ TCID₅₀). This was done by absorbing the viruscontained in 2 ml MEM media for 1 hour and then adding 17 ml mediawithout serum. At 7 days postinfection the media was removed and thecells scraped off the flasks into ice-cold PBS. Cell pellets prepared bycentrifugation at 1500 g for 5 minutes, were extracted using "RNAIsolator" (Genosys). Typically, cells from 2 flasks were extracted with1 ml extraction reagent.

(b) Extracellular virus RNA

Supernatant medium (usually 700-900 ml volumes) from infected CHSE cellswas concentrated 5 or 6 fold by Amicon filtration andultracentrifugation at 35000 rpm for 4 hours using the Beckman 8×70 mlfixed angle rotor to produce crude virus pellets, which were shown tocontain high titres of virus infectivity. Typically crude virus pelletsderived from 300-450 ml supernatant were extracted with 1 ml "RNAIsolator" reagent.

Transfection

RNA pellets were centrifuged after storage in EtOH at -20° C. at 12000 gfor 10 minutes and dissolved in 20 μl RNAase free water T.ypically,Intracellular RNA derived from the cells contained in approximately 0.25flask monolayer and Extracellular virus RNA derived from 50-100 mlsupernatant were used. Transfection was performed with the "Lipofectin"(Gibco BRL) reagent using the method recommended by the manufacturers.Briefly, this involved mixing RNA solutions with tissue culture mediacontaining Lipofectin and then incubating semiconfluent CHSE cells,grown in 24 well Costar plates, with the mixture (100 μl/1.5 cm diameterwell culture) for 5 to 6 hours before replacing the reagent transfectionmixture with maintenance medium. Cultures were examined for cpe at dailyintervals. Occasionally viral cpe was observed after 4 to 6 daysfollowing the subculture of transfected cultures, the subculturingprocedure usually being performed 8 days after the transfection.

We claim:
 1. As isolated Fish Pancreas Disease Virus (FPDV) that causesPancreas Disease (PD) in fish.
 2. The virus as claimed in claim 1, whichis the strain deposited at ECACC under Deposit No. V94090731.
 3. Thevirus as claimed in claim 1, which is free of other viral or microbialmaterial.
 4. A vaccine to protect fish against Pancreas Disease, saidvaccine comprising an inactivated virus as claimed in claim
 1. 5. Thevirus of claim 1, which is inactivated.
 6. An isolated antibody thatwill bind selectively to a virus as claimed in claim 1 or to a componentthereof.
 7. The antibody as claimed in claim 6 having a detectablemarker.
 8. The antibody as claimed in claim 6, which is in immobilizedform.
 9. A method of diagnosing Fish Pancreas Disease in a fish, saidmethod comprising the following steps:1) contacting a test sample with adiagnostic reagent as claimed in claim 6 to produce a reagent complex;and 2) determining the presence or the concentration of reagent complexand thus the presence or amount of virus in the test sample.
 10. Amethod as claimed in claim 9, wherein the test sample is a blood or asample of the water in which the fish has been contained.
 11. The methodof claim 9, further comprising a washing step after step 1) and beforestep 2).
 12. A method of isolating a virus as claimed in claim 1, saidmethod comprising identifying fish suffering from Pancreas Disease,co-cultivating affected tissues with Chinook salmon embryo cells;passaging the co-cultivated cells through Chinook salmon embryo cells;and isolating the virus particles.
 13. A method as claimed in claim 12in which the affected tissues are the pancreas or kidney, andco-cultivation with Chinook salmon embryo cells is undertaken for aperiod of approximately 28 days.